CN102212621A - Detection kit and detection method for hepatitis B virus genotyping - Google Patents
Detection kit and detection method for hepatitis B virus genotyping Download PDFInfo
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Abstract
The invention discloses a detection kit and a detection method for hepatitis B virus genotyping. The kit comprises a primer pair for polymerase chain reaction (PCR) amplification of a hepatitis B virus gene S area and a PreS2 area, a solid phase detection array, and a nucleic acid amplification reaction system and a color development reagent on a solid phase carrier containing DNA polymerase and reaction substrate, wherein a type detection probe and a contrast probe are fixed on the surface of the solid phase detection array. According to the gene chip-based high-flux, high-specificity, quick and easily-popularized detection method using a PCR technology as the principle, the detection probe is fixed on the detection array and hybridized with a PCR amplified gene fragment to be detected; and after the target fragment is identified by the probe on the solid phase carrier, a nucleotide sequence can be synthesized and prolonged, so that the biotin-labeled amplified fragment is fixed on the detection array, is not eluted and further used for color development and display, and the gene type of the hepatitis B virus to be detected can be detected at one time.
Description
Technical field
The invention belongs to field of medical examination, the somatotype that relates to hepatitis B virogene detects particularly a kind of detection kit and detection method that is used for hepatitis B virus gene typing.
Background technology
Hepatitis B is a kind of worldwide disease that is caused by hepatitis B virus (HBV).Developing country's sickness rate height, it is about 9,300 ten thousand that hepatitis B hepatitis virus carrier of China and patient amount to, wherein 1/3 clinical manifestation that hepatic injury occurs.There is hepatitis B patient 3,000 ten thousand in China at present.After the HBV infection, can develop into even more serious hepatopathy gradually, as liver cirrhosis, liver cancer etc., the death toll that causes thus reaches 500,000 every year.
In recent years studies show that the different HBV genotype that causes by transgenation, be the distribution of geographic area property, and the pathogenic difference of different genotype, the progress of the genotype and the hepatitis B state of an illness, clinical manifestation, treatment resistance, prognosis have confidential relation.Yet lack at present effectively and accurate test method comes HBV is carried out early stage detection and somatotype, this technical disappearance has brought many inconvenience for early treatment and the postoperative inspection of HBV.
Present hepatitis B classifying method has a lot, mainly is the inspection at the caused difference of transgenation.Current classifying method comprises full gene sequencing, surface antigen gene (S gene) sequential analysis, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) gene type, monoclonal antibody enzyme linked immunosorbent assay gene type method, Auele Specific Primer-PCR method, PCR microplate nucleic acid hybridization-ELISA method, linear probe analysis (LiPA) gene type method, real-time quantitative PCR one curve analysis (Real-time PCR and melting curve analysis).But these methods are for medical system, and all various degrees is difficult to popularization, cost costliness and long problem of cycle.
So, in present medical system,, seldom adopt somatotype to detect to only doing infectious the inspection under the hepatitis B patient routine, all very unfavorable to this sick early diagnosis, prognosis inspection and personalized medicine.
Gene chip is as having concurrently fast and the testing tool of the high advantage of flux of occurring in recent years, combine the order-checking principle, be by fixing tool nucleic acid probe or protein targetedly on solid phase carrier, and then combine with detected components hybridization, the back is introduced luminous signal (as fluorescence, vitamin H, radio isotope etc.) and is carried out analyzing and testing and come analytic sample.But the nucleic acid hybridization chip is equipped with equipment complexity, costliness at present, has false negative, false positive rate simultaneously, so the quality control of nucleic acid molecule gene chip is difficult to be grasped in addition.
The solid phase round pcr is to utilize the PCR principle, and the Auele Specific Primer as kapillary, silica gel sheet, magnetic bead, the first-class fixedly modification of sheet glass on solid phase carrier carries out the PCR reaction, is characterized in that reaction is simple, the accuracy height.But with regard to actually operating, modify and the fixing comparatively complex chemical treatment that needs for primer, as on silica gel sheet or kapillary, not only needing carrier surface is handled, need simultaneously probe or primer are modified, and can not apply with the supporting PCR instrument of solid phase carrier, so not only make and the production process complexity cause that simultaneously experimental repeatability reduces, and causes conventional solid phase PCR to finish scientific effort under laboratory environment.
Summary of the invention
The technical problem that the present invention solves is to provide a kind of detection kit and detection method that is used for hepatitis B virus gene typing, is that the basis is carried out flux and detected with the gene chip, has realized high specific and fast gene type and the sudden change of hepatitis B virus detected.
The present invention is achieved through the following technical solutions:
A kind of detection kit that is used for hepatitis B virus gene typing, comprise the primer that is used for pcr amplification hepatitis B virogene S district and PreS2 district to, surface be fixed with somatotype detection probes and contrast probe the solid phase detection arrays, comprise nucleic acid amplification reaction system and colouring reagents on the solid phase carrier of archaeal dna polymerase and reaction substrate;
The right 5` end of described primer increases biotin labeling;
Described somatotype detection probes comprises the detection probes at one or more genotype hepatitis B viruses, its length is 15～25nt, wherein be arranged on after the probe 5` end 5nt, and reduce sterically hindered sequence at detection probes 5` end and prolong and modify and/or auxiliary fixing the modification at the site of detecting mutation polymorphism;
Described colouring reagents comprises the compatible reaction liquid that contains avidin-alkaline phosphatase and contains the color reaction liquid of NBT/BCIP.
The fragment length that described amplimer increased is no more than 1000bp; On detection probes, also add the modification that PNA, LNA or sulfenyl improve detection specificity.
Sequence at the somatotype detection probes of A type hepatitis B virus is SEQ ID NO:1～SEQ IDNO:2;
Sequence at the somatotype detection probes of Type B hepatitis B virus is SEQ ID NO:3～SEQ IDNO:4;
Sequence at the somatotype detection probes of C type hepatitis B virus is SEQ ID NO:5～SEQ IDNO:6;
Sequence at the somatotype detection probes of D type hepatitis B virus is SEQ ID NO:7～SEQ IDNO:8;
Sequence at the somatotype detection probes of E type hepatitis B virus is SEQ ID NO:9～SEQ IDNO:10;
Sequence at the somatotype detection probes of F type hepatitis B virus is SEQ ID NO:11～SEQID NO:12;
Sequence at the somatotype detection probes of G type hepatitis B virus is SEQ ID NO:13～SEQID NO:14;
Sequence at the somatotype detection probes of H type hepatitis B virus is SEQ ID NO:15～SEQID NO:16.
Described contrast probe comprises negative control probe and positive control probe, and its designed sequence does not all have complementarity with sequence to be detected, and the 5` end of positive control probe is also by biotin labeling.
Describedly reduce sterically hindered sequence and prolong that to modify be that prolongation in that somatotype detection probes 5` end carries out polyT, polyC, polyT+polyC or spacer 6～15C sequence is modified;
The carrier of described solid phase detection arrays is nitrocellulose filter, nylon membrane or slide glass, and auxiliary fixedly being modified at somatotype detection probes 5` least significant end of somatotype detection probes 5` end added amino, sulfydryl, hydroxyl or aldehyde radical.
Described avidin reaction solution consists of 1mL ddH
2O and 2 μ l avidin-alkaline phosphatase liquid; Color reaction liquid consists of 1mL colour developing damping fluid, 6.5 μ lNBT and 3.5 μ l BCIP.
A kind of detection method of the hepatitis B virus gene typing based on the mentioned reagent box may further comprise the steps:
1) is template with the DNA that comprises hepatitis B virogene group to be measured, to being amplimer, carries out a plurality of round-robin pcr amplifications, obtain pcr amplification product with primer;
2) pcr amplification product is carried out denaturing treatment after, place nucleic acid amplification reaction system on the solid phase carrier, and then add the solid phase detection arrays, carry out the solid-phase nucleic acid amplification reaction; Its response procedures is: A: 10s at least anneals under the temperature that is not less than 5 ℃ of probe Tm values; B:72 ℃ is extended 10s at least; The A-B cycle number is at least greater than 1; Last 72 ℃ are extended 3min at least;
3) after solid-phase nucleic acid amplification is finished, take out the solid phase detection arrays and put into the centrifugal wash-out of elutriant, the combination of removing non-specific nucleic acid with compatible reaction liquid covering solid phase detection arrays and hatch, is used ddH behind the wash-out once more
2The O flushing covers the color reaction liquid that comprises NBT and BCIP at last and develops the color, and uses ddH after seeing the colour developing spot
2The O termination reaction;
6) colour developing spot pairing somatotype result is the genotype of the hepatitis B virus that is detected, according to colour developing spot and somatotype detection probes at genotype, the genotype of judgement hepatitis B virus.
Described solid phase detection arrays before use also volumetric concentration be among 2%～5% the BSA 37 ℃ seal 15min at least.
Described denaturing treatment is that thermally denature is handled: place 5min more than 95 ℃ at least, place 3min at least at 0 ℃ again;
Perhaps, described denaturing treatment is that alkaline denaturation is handled: the NaOH solution of mass concentration 10% is mixed according to 1: 10～20 volume ratio with pcr amplification product, place 3min at least.
After the reaction of described solid-phase nucleic acid amplification is finished, take out the solid phase detection arrays and at first put into elutriant A, rotating speed is the centrifugal wash-out of 30rpm 1min at least at least; Then the solid phase detection arrays is covered behind the compatible reaction liquid 37 ℃ hatch at least 10min after, put into elutriant A, elutriant B more successively, rotating speed is the centrifugal wash-out of 30rpm 1min at least at least; Take out solid phase detection arrays ddH
2O flushing covers color reaction liquid at last and develops the color, to seeing termination reaction behind the colour developing spot;
The Tris-Cl that consists of 100mmol/L pH=7.5 of described elutriant A, 300mmol/L NaCl and 4mmol/L Mg
2Cl; The Tris-Cl that consists of 100mmol/L pH=9.5 of elutriant B, 300mmol/L NaCl and 4mmol/L Mg
2Cl.
Compared with prior art, the present invention has following beneficial technical effects:
1, the detection that is used for hepatitis B virus gene typing provided by the invention, be based on the detection in Gene Mutation of nucleic acid amplification on the solid phase carrier, detection probes is fixed on the solid phase detection arrays, hybridize with the testing gene fragment of pcr amplification, after the detected probe identification of the fragment of amplification, can carry out the prolongation (solid-phase nucleic acid amplification) of sequence, make and to be fixed on the membrane array by biotin labeled amplified fragments and not by wash-out, and the sequence that can not be discerned by probe, just can not carry out the prolongation of sequence, can not be fixed by detection probes, and then be washed away; Like this by designing the distribution that a plurality of detection probes are matrix form, by once just detecting the emergent properties of gene locus to be detected.
The present invention is by based on gene chip, PCR reaction technology being the high-throughput, high specific of principle, fast and the detection method that is easy to promote, by on the solid-phase media that with the nitrocellulose filter is representative, carrying out the nucleic acid amplification reaction that specific probe causes, in conjunction with reverse dot blot hybridization technique, after will carrying biotin labeled target sequence and probe on the membrane array combining, by biotin-avidin-alkaline phosphatase-BCIP/NBT reaction, can observe obvious bluish voilet spot, thereby carry out the naked eyes interpretation.
2, the detection of hepatitis B virus gene typing provided by the invention, the detection accuracy height: comprise by specific probe design, utilize ripe round pcr annealing temperature to stop non-specific combination and to utilize Taq enzyme 5`-3` 5 prime excision enzyme activity to stop extension after the mispairing, can reach good discrimination power fully, ensure the accuracy that detects nucleotide sequence list base difference;
And less demanding to laboratory and plant and instrument: on common lab PCR instrument, can finish integral experiment, need not special and expensive device; Simple to operate; Mainly operating with the round pcr is core, and routine operation is with low cost, is easy to promote.
3, the detection of hepatitis B virus gene typing provided by the invention, carry out at hepatitis B virogene S district and PreS2 district, in the HBV genome, has the highest conservative property because compare this gene regions, so selected the interior conserved sequence of 8 genotypic types of HBV as purpose fragment to be measured according to bioinformatic analysis.Accuracy in order to guarantee to detect in addition, each genotype has designed 2 probes.Its detected result is reliable, accurate; And slow, the technical problems such as resolving power is not high, necessary instrument is expensive, testing process complexity of the detection speed that has solved existing method, can improve significantly only to have now hepatitis B patient is done the infectious present situation that somatotype detects of checking and seldom consider.
4, the detection of hepatitis B virus gene typing provided by the invention, the somatotype detection probes that is provided can flexible combination, to adapt to different detection crowd and testing goal, such as A, B, C, D and E, F, G, eight genotype of H are designed to 2 other chip arrays of branch, help the flexible Application of Clinical Laboratory.Because the HBV genotype of China domestic discovery accounts for the overwhelming majority with A, B, C, D, E, F, G, H genotype mostly are external input.
Description of drawings
The program synoptic diagram that Fig. 1 combines, extends, develops the color for amplified fragments and somatotype detection probes on being fixed on solid phase carrier; Wherein 1. being nitrocellulose filter, for the 5` end prolongs the C10 chain of modifying, 3. is the somatotype detection probes 2., 4. is amplified fragments target complement sequence district, 5. is the Taq enzyme, 6. is biotin labeling, 7. is avidin-alkaline phosphatase, 8. is the BCIP/NBT colour developing;
Fig. 2 is the specificity authentication chip array distribution synoptic diagram of hepatitis B virus typing probes;
Fig. 3 detects with the chip array distribution schematic diagram of detection probes with the contrast probe for the hepatitis B virus somatotype;
Fig. 4 is that the specificity of hepatitis B virus C type typing probes is identified colour developing figure as a result;
The colour developing that Fig. 5 detects for the hepatitis B virus somatotype is figure as a result; Fig. 5 a～Fig. 5 d is respectively the colour developing figure as a result of A type, Type B, C type, the D type of detected HBV, and Fig. 5 e～Fig. 5 h is respectively the colour developing figure as a result of E type, F type, G type, the H type of detected HBV.
Embodiment
Below in conjunction with the design that detects segmental amplification and detection probes in the concrete hepatitis B virus somatotype detection, fixing of detection probes and solid phase carrier, detection probes and amplified fragments are hybridized on solid phase carrier, are increased, the present invention is described in further detail, and the explanation of the invention is not limited.
Referring to Fig. 1, detection method of gene mutation based on nucleic acid amplification on the solid phase carrier, a kind of detection kit that is used for hepatitis B virus gene typing that is provided, comprise the primer that is used for pcr amplification hepatitis B virogene S district and PreS2 district to, surface be fixed with somatotype detection probes and contrast probe the solid phase detection arrays, comprise nucleic acid amplification reaction system and colouring reagents on the solid phase carrier of archaeal dna polymerase and reaction substrate.
It is right to utilize ClustalX and Bioedit software to carry out that 47 sequences of 8 types of hepatitis B viruses (HBV) of having reported are carried out multiple ratio, the interior conserved sequence of type of finding out S district and PreS2 district is as purpose fragment to be measured, common PCR amplification in vitro is adopted in its amplification, require cycle number 25～40, the length suggestion of amplified fragments is no more than 1000bp, and amplimer both can also can have by deciding in the position of amplified fragments relation in mutational site to be measured on reverse primer at forward primer the mark of last avidin; The Design of length of detection probes is 15～25nt, is arranged on after the 5` end 5nt at the site of detecting mutation polymorphism on the detection probes.
Described somatotype detection probes comprises the detection probes at one or more genotype hepatitis B viruses, can be to comprise all, the genotypic specific sequence site of site for detecting of underscore mark wherein arranged at known A～G totally 8 kinds of genotypic somatotype detection probes;
Sequence at the somatotype detection probes of A type hepatitis B virus is SEQ ID NO:1～SEQ IDNO:2;
Sequence at the somatotype detection probes of Type B hepatitis B virus is SEQ ID NO:3～SEQ IDNO:4;
Sequence at the somatotype detection probes of C type hepatitis B virus is SEQ ID NO:5～SEQ IDNO:6;
Sequence at the somatotype detection probes of D type hepatitis B virus is SEQ ID NO:7～SEQ IDNO:8;
Sequence at the somatotype detection probes of E type hepatitis B virus is SEQ ID NO:9～SEQ IDNO:10;
Sequence at the somatotype detection probes of F type hepatitis B virus is SEQ ID NO:11～SEQID NO:12;
Sequence at the somatotype detection probes of G type hepatitis B virus is SEQ ID NO:13～SEQID NO:14;
Sequence at the somatotype detection probes of H type hepatitis B virus is SEQ ID NO:15～SEQID NO:16;
And add the modification of polyT (10)+polyC (10) at its 5` end.
In the somatotype detection probes that is provided, each genotypic two kinds of detection probes can be used separately, is fixed on the same distribution sites after also two kinds of probes can being mixed.
Consider that the HBV genotype is the distribution of geographic area property, concrete genotypic selection can according at the detection crowd, carry out suitable screening; The patient who infects E, F, G, H type HBV such as China is extremely rare, the probe array commonly used that can directly be distributed with A, B, C, the detection of D four types detects first, when needed, the detection probes that is distributed with E, F, G, the detection of H four types detects once more; Also can with A～H totally 8 kinds somatotype detection probes set be distributed on the array and carry out one-time detection.
The contrast probe comprises negative control probe and positive control probe, and the sequences Design of negative control probe and positive control probe can be identical or inequality, and principle is that its sequence can not have cross complementary with amplified fragments.Whether the 5` end of positive control probe can be developed the color by biotin labeling, normal in order to the monitoring Color Appearance System; Negative control probe is in order to the specificity of monitoring hybridization and solid-phase nucleic acid amplification.
The sequence of concrete contrast probe can be designed as:
Positive control sequence B iotin-acagcactaa ggcaagctat tctg;
Negative control sequence acagcactaa ggcaagctat tctg.
The modification of somatotype detection probes 5` end comprises: reduce sterically hindered sequence and prolong modification and/or auxiliary fixing the modification; Wherein reducing sterically hindered sequence prolongation modification is for the sequence after reducing the nucleic acid amplification mispairing and increasing and the consideration of adhering to, developing the color of solid phase carrier, comprises the prolongation modification of multiple sequences such as polyT, polyC, polyT+polyC or spacer 6～15C.
5` end auxiliary fixing modifies general and solid phase carrier is taken into consideration, solid phase carrier can be nitrocellulose filter, nylon membrane or slide glass, fix with it by auxiliary fixing modification of adding amino, sulfydryl, hydroxyl or aldehyde radical at the 5` of detection probes end, the better fixing and color developing effect in the future for detection probes, solid phase carrier can carry out kinds of surface and modify.
Such as, be solid phase carrier with the nitrocellulose filter, after drying again by immersion in SSC solution, can be so that the indiffusion of colour developing spot; By the hydroxyl of detection probes 5` end, behind ultraviolet lighting, fix with nitrocellulose filter.
Modification for probe specificity can be used methods such as PNA, LNA, sulfydryl, increases the Tm value of probe or the specificity of also extending by the change raising probe combination of polysaccharase space conformation.
Solid-phase nucleic acid amplification is that detection probes and target gene fragment are hybridized on solid phase carrier, increased, it is the core of probe specificity identification, wherein, amplification system adopts conventional PCR reaction solution, its unique distinction just is with the somatotype detection probes that comprises detection site as primer, can and carry out nucleic acid amplification with the complementary hybridization of goal gene, and be fixed on the solid phase carrier not by wash-out by detection probes; Simultaneously, for the specificity that guarantees to hybridize, the annealing temperature operated by rotary motion is in being not less than 5 ℃ of scopes of probe Tm value.
Elution process is in order will not wash away with solid phase carrier bonded amplified fragments, and elutriant can be for general damping fluid commonly used, and is relatively good in conjunction with centrifugal elute effect.
Colour developing it is advantageous that by the display mode of avidin-alkaline phosphatase liquid+NBT/BCIP background color is not obvious, and reaction is accurate, and the result judges that easily effect is relatively good; And realize that conveniently avidin-alkaline phosphatase liquid and NBT/BCIP colour developing liquid all can adopt existing test kit.
Below in conjunction with the genotype tests process of hepatitis B virus, test kit and detection method thereof are carried out specific description:
1) detects the preparation of using amplified production
Design of amplification primers is right, and the 5` end of reverse amplimer is increased vitamin H (Biotin) mark, and designed primer is specially P:
Forward primer: ctgctggtgg ctccagtt 18;
Reverse primer: Biotin-gcactagtaa actgagcca 19;
Utilizing virus to extract the genomic dna that test kit extracts hepatitis B viruses (HBV) from hepatitis B patient serum, is template with the genomic dna, is that primer carries out 30 round-robin pcr amplifications with primer to P, can obtain the amplified fragments that length is 630bp.
2) design of detection probes is with synthetic
Design and synthetic eight kinds of somatotype detection probes of A～G of hepatitis B viruses (HBV), 2 probes of every type, probe Tm value is designed to 59 ± 1 ℃, probe length is 16～24nt, holds at probe sequence 5` simultaneously to add polyT (T
10), polyC (C
10) sequence prolong to modify, reducing that sterically hindered prolongation modifies is in order to be beneficial to target sequence and to be fixed on epilamellar probe and to combine, concrete detection probes sequence is shown in SEQ ID NO:1～SEQ ID NO:16, can select every kind of genotype at probe a kind of, also can be two kinds of mixing, distribute together during point sample;
The site of detecting polymorphism on the probe is provided with all after probe 5` end 5nt.
3) detection probes fixing on detection arrays
With nitrocellulose filter at 2 * SSC solution (trisodium citrate 15mM, sodium-chlor 150mM, pH7.0) take out after soaking 30min in, oven dry is 2 hours in 37 ℃ of incubators, again somatotype detection probes and contrast probe are sprayed into (1 500nl on the film with some film instrument, interval 1mm, be matrix distribution), reduce to 4mm * 6mm size, the nitrocellulose filter of having put probe was put into 80 ℃ of oven for drying 30 minutes, 254nm wavelength ultraviolet lighting 6 minutes is fixedlyed connected probe by ultraviolet lighting with nitrocellulose filter, be fixed the solid phase detection arrays of detection probes and contrast probe.Under drying conditions, the solid phase detection arrays can be laid in stand-by for a long time, before using this film is immersed 2%BSA liquid and seals 15min for 37 ℃.
In order to detect of specific detection, the demonstration of somatotype detection probes, design following two kinds of array distribution to different genotype:
In order to detect the specificity that typing probes is identified, specifically illustrate as detecting with B, C type probe, its probe distribution as shown in Figure 2, it wherein 1. is the equal proportion mixture of two kinds of probes (B1 and B2) of Type B probe, 2. 3.～4. positive contrast probe is respectively the probe one and the probe two (C1, C2) of C type;
In order to detect specificity, the accuracy that every kind of typing probes is identified, design probe distribution as shown in Figure 3: be the contrast probe wherein 1., 2., 3.～6. be respectively the somatotype detection probes; And done the differentiation of array distribution with the common genotype of CHINESE REGION: the detection probes of the A type of 3.～6. being respectively of array one, Type B, C type, D type at HBV; The detection probes of the E type of 3.～6. being respectively of array two, F type, G type, H type at HBV; Each site all comprises the equal proportion mixture of two probes of homotype.
4) solid-phase nucleic acid amplification reaction
At first the gene amplification product that the first step is obtained carries out the sequence number mark, then amplified production is carried out thermally denature and handles: 95 ℃ of water-bath 5min, place 3min then on ice; Again with the 10 μ l of the amplified production after the sex change, PCR reaction solution (glad hundred promise companies, TagMixMaster) 20 μ l, ddH
2O 10 μ l put into clean PCR reaction tubes mixing, and the detection arrays for preparing is put into, and the film back side tube wall of solid phase detection arrays carries out the solid-phase nucleic acid amplification reaction; Design response procedures: A:57 ℃ of annealing 30s; B:72 ℃ is extended 30s; The A-B cycle number is 10; Last 72 ℃ are extended 3min.
Wherein the specificity of typing probes identifies it is that genomic amplified production with amplification C type hepatitis B virus is as detected object;
What array one detected is to comprise a kind of genomic amplified production of A～D type hepatitis B virus respectively as detected object;
What array two detected is to comprise a kind of genomic amplified production of F～H type hepatitis B virus respectively as detected object.
5) wash-out and colour developing
After solid phase PCR reaction is finished, take out detection arrays and carry out wash-out, will not wash away with detection arrays fixed fragment, all elution process is all finished under 37 ℃ of conditions, is specially:
Detection arrays is taken out, put into elutriant A (Tris-Cl of 100mmol/L pH=7.5,300mmol/L NaCl and 4mmol/L Mg
2Cl) centrifugal wash-out in (rotating speed is 30rpm, wash-out 1min) is after the taking-up detection arrays, with compatible reaction liquid (the 1mL ddH of alkaline phosphate ester enzyme labelling
2O and 2 μ l avidin-alkaline phosphatase liquid, available from green skies company, alkaline phosphatase is labeled as Streptavidin) cover after, hatch 10min under 37 ℃ of conditions; Hatch and after finishing detection arrays is put into clean elutriant A and carry out wash-out (rotating speed 30rpm, wash-out 1min), put into elutriant B (Tris-Cl of 100mmol/LpH=9.5,300mmol/L NaCl and 4mmol/L Mg after the taking-up again
2Cl) in, centrifugal again wash-out (rotating speed 30rpm, wash-out 1min); Take out detection arrays, with clear water to the surface washing of film piece, and utilize thieving paper to remove liquid on the film piece, the film piece is covered color reaction liquid (1mL develop the color damping fluid, 6.5 μ lNBT and 3.5 μ l BCIP, available from green skies company, BCIP/NBT alkaline phosphatase colouring reagents box), after naked eyes can be seen spot after 30 seconds, promptly use the clear water stopped reaction.
6) gene type assay
The specificity of hepatitis B virus C type typing probes is identified colour developing, and figure is as shown in Figure 4 as a result, wherein 2. normally colour developing of positive control probe, and comprise the 1. not colour developing of Type B probe B1 and probe B2, comprise C type probe C1 and probe C2 3., 4. all specific demonstration, this shows the specificity of somatotype detection probes, every kind of genotypic probe is all at the specific colour developing of the genotype of its detection, other detection probes no cross reaction: all specific colour developing of two probe site of C type, and the Type B probe there is no colour developing.And the probe to A, B, D has carried out the specificity evaluation that similar Fig. 2 designed probe distributes, and shows that all specificity is very good.
The detected result of array one is shown in Fig. 5 a～Fig. 5 d, be respectively the colour developing figure as a result of A type, Type B, C type, the D type of detected HBV, wherein 1. positive contrast, 2. negative contrast, can see by all normally colour developings of biotin labeled positive control, illustrate that Color Appearance System is working properly; Negative control does not develop the color, and illustrates that the specificity of solid-phase amplification is good, does not have non-specific hybridization and nucleic acid amplification; Have only 3. among Fig. 5 a～Fig. 5 d, 4., 5., 6. site fixed detection probes respectively with HBV virus target sequence specific combination and colour developing, has only a kind of probe in the middle of the array at certain type hepatitis B virus colour developing to be measured, carry out result's indication, other probes there is no colour developing in the membrane array; This shows that these four kinds of genotypic probes can specificly identify the genotype of hepatitis B gene group to be measured, and its result accurately and reliably, amixia dyeing each other.
And the detected result of array two is shown in Fig. 5 e～Fig. 5 h, be respectively the colour developing figure as a result of E type, F type, G type, the H type of detected HBV, wherein 1. negative contrast, 2. positive contrast, can see by all normally colour developings of biotin labeled positive control, illustrate that Color Appearance System is working properly; Negative control does not develop the color, and illustrates that the specificity of solid-phase amplification is good, does not have non-specific hybridization and nucleic acid amplification; Have only 3. among Fig. 5 a～Fig. 5 d, 4., 5., 6. site fixed detection probes respectively with HBV virus target sequence specific combination and colour developing, has only a kind of probe in the middle of the array at certain type hepatitis B virus colour developing to be measured, carry out result's indication, other probes there is no colour developing in the membrane array; This shows that these four kinds of genotypic probes can specificly identify the genotype of hepatitis B gene group to be measured, and its result accurately and reliably, amixia dyeing each other.
Detected result in conjunction with above array, after somatotype detection probes provided by the present invention is fixed and forms the detection arrays of array distribution, every kind of detection probes all can be carried out result's indication at certain type hepatitis B virus colour developing to be measured, its colour developing obviously, the result accurately and reliably.
Annotate: infect the very rare genotype of HBV for Chinese population, in the middle of the sample that the patient extracts, do not obtain virus strain as yet,, in array detection, used artificial-synthetic DNA's template in order to verify the detection effect of probe.
Accuracy and repeatability textual criticism:
Serum HBV amplified production to 100 routine Chinese hepatitis B virus patients checks order, genotyping tool with NCBI carries out somatotype, wherein the A type is artificial synthesized sequence 1 example, Type B 25 examples, C type 67 examples, D type 7 examples are compared with above-mentioned method detected result again, and it is 99% that this method detects accuracy rate.Wherein 1 routine flase drop causes because of producing sudden change in the next door, site of designing probe base.
Claims (10)
1. detection kit that is used for hepatitis B virus gene typing, it is characterized in that, comprise the primer that is used for pcr amplification hepatitis B virogene S district and PreS2 district to, surface be fixed with somatotype detection probes and contrast probe the solid phase detection arrays, comprise nucleic acid amplification reaction system and colouring reagents on the solid phase carrier of archaeal dna polymerase and reaction substrate;
The right 5` end of described primer increases biotin labeling;
Described somatotype detection probes comprises the detection probes at one or more genotype hepatitis B viruses, its length is 15～25nt, wherein be arranged on after the probe 5` end 5nt, and reduce sterically hindered sequence at detection probes 5` end and prolong and modify and/or auxiliary fixing the modification at the site of detecting mutation polymorphism;
Described colouring reagents comprises the compatible reaction liquid that contains avidin-alkaline phosphatase and contains the color reaction liquid of NBT/BCIP.
2. the detection kit that is used for hepatitis B virus gene typing as claimed in claim 1 is characterized in that the fragment length that described amplimer increased is no more than 1000bp; On detection probes, also add the modification that PNA, LNA or sulfenyl improve detection specificity.
3. the detection kit that is used for hepatitis B virus gene typing as claimed in claim 1 is characterized in that, is SEQ ID NO:1～SEQ ID NO:2 at the sequence of the somatotype detection probes of A type hepatitis B virus;
Sequence at the somatotype detection probes of Type B hepatitis B virus is SEQ ID NO:3～SEQ IDNO:4;
Sequence at the somatotype detection probes of C type hepatitis B virus is SEQ ID NO:5～SEQ IDNO:6;
Sequence at the somatotype detection probes of D type hepatitis B virus is SEQ ID NO:7～SEQ IDNO:8;
Sequence at the somatotype detection probes of E type hepatitis B virus is SEQ ID NO:9～SEQ IDNO:10;
Sequence at the somatotype detection probes of F type hepatitis B virus is SEQ ID NO:11～SEQID NO:12;
Sequence at the somatotype detection probes of G type hepatitis B virus is SEQ ID NO:13～SEQID NO:14;
Sequence at the somatotype detection probes of H type hepatitis B virus is SEQ ID NO:15～SEQID NO:16.
4. the detection kit that is used for hepatitis B virus gene typing as claimed in claim 1, it is characterized in that, described contrast probe comprises negative control probe and positive control probe, its designed sequence does not all have complementarity with sequence to be detected, and the 5` end of positive control probe is also by biotin labeling.
5. the detection kit that is used for hepatitis B virus gene typing as claimed in claim 1, it is characterized in that, describedly reduce sterically hindered sequence and prolong that to modify be that prolongation in that somatotype detection probes 5` end carries out polyT, polyC, polyT+polyC or spacer 6～15C sequence is modified;
The carrier of described solid phase detection arrays is nitrocellulose filter, nylon membrane or slide glass, and auxiliary fixedly being modified at somatotype detection probes 5` least significant end of somatotype detection probes 5` end added amino, sulfydryl, hydroxyl or aldehyde radical.
6. the detection kit that is used for hepatitis B virus gene typing as claimed in claim 1 is characterized in that, described compatible reaction liquid consists of 1mL ddH
2O and 2 μ l avidin-alkaline phosphatase liquid; Color reaction liquid consists of 1mL colour developing damping fluid, 6.5 μ lNBT and 3.5 μ l BCIP.
7. the detection method based on the hepatitis B virus gene typing of the described detection kit of claim 1 is characterized in that, may further comprise the steps:
1) is template with the DNA that comprises hepatitis B virogene group to be measured, to being amplimer, carries out a plurality of round-robin pcr amplifications, obtain pcr amplification product with primer;
2) pcr amplification product is carried out denaturing treatment after, place nucleic acid amplification reaction system on the solid phase carrier, and then add the solid phase detection arrays, carry out the solid-phase nucleic acid amplification reaction; Its response procedures is: A: 10s at least anneals under the temperature that is not less than 5 ℃ of probe Tm values; B:72 ℃ is extended 10s at least; The A-B cycle number is at least greater than 1; Last 72 ℃ are extended 3min at least;
3) after solid-phase nucleic acid amplification is finished, take out the solid phase detection arrays and put into the centrifugal wash-out of elutriant, the combination of removing non-specific nucleic acid with compatible reaction liquid covering solid phase detection arrays and hatch, is used ddH behind the wash-out once more
2The O flushing covers the color reaction liquid that comprises NBT and BCIP at last and develops the color, and uses ddH after seeing the colour developing spot
2The O termination reaction;
6) colour developing spot pairing somatotype result is the genotype of the hepatitis B virus that is detected, according to colour developing spot and somatotype detection probes at genotype, the genotype of judgement hepatitis B virus.
8. the detection method of hepatitis B virus gene typing as claimed in claim 7 is characterized in that, described solid phase detection arrays before use also volumetric concentration be among 2%～5% the BSA 37 ℃ seal 15min at least.
9. the detection method of hepatitis B virus gene typing as claimed in claim 7 is characterized in that, described denaturing treatment is that thermally denature is handled: place 5min more than 95 ℃ at least, place 3min at least at 0 ℃ again;
Perhaps, described denaturing treatment is that alkaline denaturation is handled: the NaOH solution of mass concentration 10% is mixed according to 1: 10～20 volume ratio with pcr amplification product, place 3min at least.
10. the detection method of hepatitis B virus gene typing as claimed in claim 7 is characterized in that, after the reaction of described solid-phase nucleic acid amplification is finished, takes out the solid phase detection arrays and at first puts into elutriant A, and rotating speed is the centrifugal wash-out of 30rpm 1min at least at least; Then the solid phase detection arrays is covered behind the compatible reaction liquid 37 ℃ hatch at least 10min after, put into elutriant A, elutriant B more successively, rotating speed is the centrifugal wash-out of 30rpm 1min at least at least; Take out solid phase detection arrays ddH
2O flushing covers color reaction liquid at last and develops the color, to seeing termination reaction behind the colour developing spot;
The Tris-Cl that consists of 100mmol/L pH=7.5 of described elutriant A, 300mmol/L NaCl and 4mmol/L Mg
2Cl; The Tris-Cl that consists of 100mmol/L pH=9.5 of elutriant B, 300mmol/L NaCl and 4mmol/L Mg
2Cl.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451319A (en) * | 2013-08-22 | 2013-12-18 | 中国人民解放军第四军医大学 | Application of HBV (hepatitis B virus) B genetype 1799G>C mutation as molecular marker, and kit |
| CN105256076A (en) * | 2015-11-20 | 2016-01-20 | 河北国际旅行卫生保健中心 | Method and kit for identifying and detecting eight gene subtypes of hepatitis B virus |
| CN107570092A (en) * | 2012-06-15 | 2018-01-12 | 伊鲁米那股份有限公司 | The dynamics of nucleic acid library excludes amplification |
| CN114250326A (en) * | 2022-02-28 | 2022-03-29 | 北京吉检医疗科技有限公司 | Kit for detecting hepatitis B virus DNA by one-tube method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1331346A (en) * | 2000-06-30 | 2002-01-16 | 中国科学院上海冶金研究所 | Process for preparing polymorphic test chip for hepatitis B virus and its application |
| CN1556223A (en) * | 2004-01-08 | 2004-12-22 | 中国科学院武汉病毒研究所 | Method of oligonuleotide join detecting gene mutation on gene chip |
| CN101402993A (en) * | 2007-11-05 | 2009-04-08 | 中国科学院武汉病毒研究所 | Parting method for hepatitis B virus gene |
-
2011
- 2011-05-09 CN CN201110118281.7A patent/CN102212621B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1331346A (en) * | 2000-06-30 | 2002-01-16 | 中国科学院上海冶金研究所 | Process for preparing polymorphic test chip for hepatitis B virus and its application |
| CN1556223A (en) * | 2004-01-08 | 2004-12-22 | 中国科学院武汉病毒研究所 | Method of oligonuleotide join detecting gene mutation on gene chip |
| CN101402993A (en) * | 2007-11-05 | 2009-04-08 | 中国科学院武汉病毒研究所 | Parting method for hepatitis B virus gene |
Non-Patent Citations (2)
| Title |
|---|
| KENICHI TADOKORO等: "Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes", 《JOURNAL OF VIROLOGICAL METHODS》, vol. 138, 24 August 2006 (2006-08-24), pages 30 - 39 * |
| 鲁雪等: "核酸扩增新技术", 《动物医学进展》, vol. 31, no. 3, 31 December 2010 (2010-12-31), pages 103 - 106 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107570092A (en) * | 2012-06-15 | 2018-01-12 | 伊鲁米那股份有限公司 | The dynamics of nucleic acid library excludes amplification |
| CN107570092B (en) * | 2012-06-15 | 2020-03-17 | 伊鲁米那股份有限公司 | Kinetic exclusion amplification of nucleic acid libraries |
| US11254976B2 (en) | 2012-06-15 | 2022-02-22 | Illumina, Inc. | Kinetic exclusion amplification of nucleic acid libraries |
| CN103451319A (en) * | 2013-08-22 | 2013-12-18 | 中国人民解放军第四军医大学 | Application of HBV (hepatitis B virus) B genetype 1799G>C mutation as molecular marker, and kit |
| CN105256076A (en) * | 2015-11-20 | 2016-01-20 | 河北国际旅行卫生保健中心 | Method and kit for identifying and detecting eight gene subtypes of hepatitis B virus |
| CN105256076B (en) * | 2015-11-20 | 2018-08-28 | 河北国际旅行卫生保健中心 | The discriminating detection method and kit of eight kinds of gene hypotypes of hepatitis type B virus |
| CN114250326A (en) * | 2022-02-28 | 2022-03-29 | 北京吉检医疗科技有限公司 | Kit for detecting hepatitis B virus DNA by one-tube method and application thereof |
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